Suppressor Cell Population Research Articles

Abstract 3271: A systemically administered, conditionally active TLR8 agonist for the treatment of HER2-expressing tumors

Abstract Clinical development of systemically administered myeloid cell agonists has been hindered by acute toxicities due to peripheral activation of the targeted cell types. Intratumoral administration, the route of delivery typically used for innate immune/myeloid cell agonists, is limited by tumor accessibility and a dependence on abscopal responses. Here, we describe an example of a new composition class, termed ImmunoTAC, that allows for systemic delivery of an immune modulator with activity localized to tumor sites and secondary lymphoid structures. Agonism of TLR8 in human myeloid cells drives anti-tumor immunity by inducing direct macrophage killing of tumor cells, repolarizing suppressive myeloid cell populations to a pro-immunogenic phenotype, and inducing dendritic cells to drive tumor-specific CTL responses. SBT6050 is an ImmunoTAC comprised of a novel, potent TLR8 agonist conjugated to a HER2-directed monoclonal antibody. SBT6050 is designed to activate human monocytes and macrophages only in the presence of HER2pos tumor cells with moderate (2+ by IHC) or high (3+ by IHC) expression levels. This activity is dependent upon the ability of the Fc domain of the antibody to engage Fcγ receptors on the surface of the myeloid cells. Additionally, SBT6050 potently activates conventional dendritic cells that, in turn, drive a Th1/CTL program in T cells. Systemic delivery of a SBT6050 surrogate in mice shows robust single agent efficacy in tumor models intrinsically resistant to checkpoint blockade, such as the previously characterized HER2+ CT26 syngeneic model. The intratumoral immune response is characterized by robust activation of tumor-associated myeloid cells, infiltration of neutrophils, persistent increases in local cytokine and chemokine production, decreases in Treg infiltrate, and the generation of a potent, neo-Ag specific anti-tumor CTL response. Mice cured with single-agent treatment are resistant to tumor rechallenge, demonstrating induction of long-lived immunological memory. In contrast to observations with small molecule TLR8 agonists, ImmunoTAC does not induce peripheral cytokine production or associated CRS-like toxicity in mice, consistent with the localized activity of the molecule. Silverback’s lead candidate, SBT6050, is currently in preclinical development for patients with moderate or high HER2-expressing tumors. More broadly, the data presented here describe a novel therapeutic modality that allows for systemic administration of immune modulators with tissue-localized activity. Citation Format: Kara Moyes, Ty Brender, Sean W. Smith, Hengyu Xu, Ben Setter, Li-Qun Fan, Rebecca Brunette, Justin Killebrew, Phil Tan, Craig Coburn, Peter Baum, Valerie Odegard. A systemically administered, conditionally active TLR8 agonist for the treatment of HER2-expressing tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3271.

Abstract CT002: A strong HPV-specific T-cell response after chemo-immunotherapy for advanced cervical cancer is associated with prolonged survival

Abstract The therapeutic synthetic long peptide (SLP) vaccine ISA101 selectively enlarges the pool of tumor-specific T cells recognizing the human papillomavirus type 16 (HPV16) antigens E6 and E7. Vaccine monotherapy for HPV16-induced pre-malignant anogenital lesions is effective, inducing durable complete regressions in approximately 50% of treated patients. However, cancer treatment requires appropriate countermeasures to overcome the suppression of T-cell activation, expansion and effector function imposed by suppressive myeloid cell populations, regulatory T cells and co-inhibitory molecule expression. We recently showed that carboplatin/paclitaxel chemotherapy can normalize the abnormally high levels of immune suppressive myeloid cells, allowing the development of much stronger therapeutic ISA101-induced tumor immunity1. Here we show the effects of ISA101 vaccination during chemotherapy in 77 patients with advanced, recurrent or metastatic cervical cancer in a dose assessment study. Study design involved a single arm dose escalation study with four different ISA101 doses (20, 40, 100 and 300 µg per peptide) with or without immunomodulator pegylated interferon alpha (Pegitron), given concomitantly with standard of care chemotherapy (carboplatin, AUC 6; paclitaxel, 175 mg/m2). Objective regressions were observed in 43% of 72 evaluable patients. The depletion of myeloid suppressive cells by carboplatin/paclitaxel was associated with detection of low level spontaneous HPV16-specific immunity in 26 of the 61 tested patients. Patients mounted type 1 T-cell responses to the vaccine across all doses. No significant differences were observed between the different doses and strength of vaccine-induced T cell responses; the results were therefore combined. Patients with an above median HPV16-specific T cell immune response to ISA101, measured by a validated IFNγ-Elispot, displayed a significantly prolonged median overall survival (OS) of 16.8 months compared to those with a lower HPV-specific immune response (OS 11.2 months, logrank p=0.012). Importantly, this effect was not due to differences in general immune status, as measured by T-cell reactivity to unrelated common microbial recall antigens. In conclusion, our study demonstrates that chemo-immunotherapy can be exploited to the benefit of patients with cervical cancer and warrants confirmation of the benefit of this type of chemo-immunotherapy in a randomized controlled study in HPV16-induced cancer patients. 1Welters, M. J. et al. Vaccination during myeloid cell depletion by cancer chemotherapy fosters robust T cell responses. Sci Transl Med 8, 334ra352, doi:10.1126/scitranslmed.aad8307 (2016). Citation Format: Cornelis J M Melief, Marij J. Welters, Ignace Vergote, Judith R. Kroep, Gemma G. Kenter, Nelleke Ottevanger, Wiebren A. Tjalma, Hannelore Denys, Mariette van Poelgeest, Hans W. Nijman, Anna K. Reyners, Thierry Velu, Frederic Goffin, Roy Lalisang, Willem-Jan Krebber, Leon Hooftman, Sonja Visscher, Brent A. Blumenstein, Richard B. Stead, Winald Gerritsen, Sjoerd van der Burg. A strong HPV-specific T-cell response after chemo-immunotherapy for advanced cervical cancer is associated with prolonged survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT002.

Abstract 5028: Characterization of the tumor immune microenvironment in treatment-naïve EGFR-mutant NSCLC uncovers a low IFN-gamma suppressive immune phenotype

Abstract Background: Although immune checkpoint blockade has been successfully utilized in treating patients with non-small cell lung cancer (NSCLC), the benefit of immunotherapy for patients with advanced EGFR-mutant (EGFRm) NSCLC has been limited. Data from IMpower150 trial’s subgroup analysis suggests modulation of the tumor immune microenvironment (TME) with antiangiogenic agents and chemotherapy might enhance response to anti-PD-1/PD-L1 blockade in EGFRm lung cancers. We aim to gain a deep understanding of EGFRm NSCLC TME as the first step to make EGFRm amenable to immune therapy. Methods: We queried two cohorts of resected stage I-III lung adenocarcinomas: PROSPECT (94 tumors) and Immune Genomic Profiling of NSCLC (ICON, 76 tumors). In the PROSPECT cohort (14 EGFRm vs 80 WT), we compared 10 immune markers by IHC (PD-L1, PD-1, CD3, CD4, CD8, CD45RO, CD57, CD68, FoxP3 and Granzyme B) and 44 immune regulator genes’ expression levels and pathway signatures from microarray data. We subsequently validated the observation of differentially expressed genes in the ICON cohort (15 EGFRm vs 61 WT) using IF, RNAseq and WES. Results: In the PROSPECT cohort, lower PD-L1 (2.2 vs 10.4 H-score, p<0.01) and lower GzmB (212 vs. 358 counts/mm2, p=0.02) were observed in the EGFRm compared to WT tumors. This is validated in the ICON cohort, showing lower PD-L1 expression (0.18% vs 7.28% p=0.05) and lower TMB (1.8 vs 9.0 mut/Mb, p<0.01) in EGFRm compared to WT tumors. IFN-gamma gene expression (3.89 vs 4.71 p=0.03 in PROSPECT) and signatures were low in the EGFRm tumors, suggesting a suppressive tumor microenvironment. Different than some prior reports, we found CD8+ T cells were not significantly different in EGFRm vs. WT groups. Among known immune regulators, TGFbeta was higher in the EGFRm tumors in the PROSPECT cohort, but not in the ICON cohort. Other known immune regulators, including CTLA4, LAG3, TIM3, TIGIT, IL6 and VEGFA were not differentially expressed. Conclusion: Our analysis showed that EGFR-mutant NSCLCs demonstrate a PD-L1 low, GzmB low, IFN-gamma low immune phenotype, suggesting a suppressive tumor microenvironment. We found that the CD8+ T cells were present in the tumor, but likely suppressed functionally by the negative regulators in the TME. These results direct future analysis of suppressive immune cell populations (CD4+ subpopulation, macrophages and dendritic cells) in EGFRm lung cancers, and represent an initial step for rationale combination of immune therapy to modulate the suppressed TME, which might lead to enhanced treatment efficacy to benefit patients with EGFR-mutant lung cancers. Citation Format: Xiuning Le, Marcelo V. Negrao, Alexandre Reuben, Won-Chul Lee, Edwin Parra, Jun Li, Tatiana Karpinets, Carmen Behrens, Boris Sepesi, Ara Vaporiciyan, Jack Roth, Cara Haymaker, Emily Roarty, Jianhua Zhang, Chantale Bernatchez, Jianjun Zhang, Ignacio Wistuba, Don Gibbons, John Heymach. Characterization of the tumor immune microenvironment in treatment-naïve EGFR-mutant NSCLC uncovers a low IFN-gamma suppressive immune phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5028.

Abstract 3948: The sequential administration of vinca alkaloids and intermittent cyclophosphamide primes antigen-presenting and NK cells and significantly improves in vivo efficacy of anti-PD-L1 in triple-negative breast cancer models

Abstract Checkpoint inhibitors (CIs) have shown an unprecedented clinical activity in a large variety of cancer types, but only in a fraction of patients. To increase the number of responders, preclinical models are studied to define effective combinatorial regimens and the best window of therapeutic opportunities. We recently described a synergy between anti-PD-1 and -PD-L1 CIs and several types and dosages of chemotherapy in immunocompetent models of triple negative breast cancer (TNBC), likely due to unique effects of chemotherapeutics over circulating and tumor-infiltrating, suppressive, antigen-presenting and effector immune cell populations (Orecchioni et al, Br J Cancer 2018). To find a high-order drug combination, we have further studied by multiparametric flow cytometry and single-cell transcriptomic a panel of different chemotherapy drugs (including microtubular poisons, alkylating agents, topoisomerase inhibitors and antimetabolites) used in vivo at different dosages and schedule. Our aim was to define a combination, dose and schedule able to promote a) dendritic cell maturation, b) release and c) uptake of cancer-associated antigens from targeted neoplastic cells, d) cross-priming between antigen-presenting cells (APCs) and T cells, e) NK cell activation, and f) inhibition of suppressor immune cell populations. Vinca alkaloids vinblastine and vinorelbine (V), at low-medium dosages, were the most effective in a), b), c) and d). After V administration, Tregs were reduced, and circulating and tumor-infiltrating APCs showed a “maturation to activation” program with increased expression of several antigens including CD40, CD80, and CD86. The alkylating agent cyclophosphamide (CTX) targeted Tregs, mobilized APC progenitors, activated and increased the number of circulating and intratumoral NK cells. These effects were significantly increased when CTX was administered in an intermittent fashion (every 6 days) at low-medium doses. As the APC-NK cell axis has been recently found to be pivotal in CI-mediated, anti-tumor cell immunity, our data suggested a preclinical trial in immunocompetent orthotopic models of metastatic (post-mastectomy) BALB mice injected with 4T1 or EMT-6 TNBC cells. Among a large panel of different combinations and dosages, the sequential administration of V, intermittent CTX and anti-PD-L1 was the only able to completely abrogate TNBC local and metastatic growth, and this effect was not observed in T and NK cell-depleted mice. Taken together with recent data from randomized, combinatorial studies in TNBC patients (Schmid et al, NEJM 2018), our findings suggest that CIs might be included in future combinatorial regimens aimed at improving the percentage of patients receiving a clinical benefit and prolonging the duration of this benefit. Citation Format: Stefania Orecchioni, Loredana Vecchi, Paolo Falvo, Lucilla Luzi, Patrizia Mancuso, Chiara Camisaschi, Francesco Bertolini. The sequential administration of vinca alkaloids and intermittent cyclophosphamide primes antigen-presenting and NK cells and significantly improves in vivo efficacy of anti-PD-L1 in triple-negative breast cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3948.

Gr-MDSC-linked asset as a potential immune biomarker in pretreated NSCLC receiving nivolumab as second-line therapy.

Immunotherapy is a new standard first-line treatment for non-small cell lung cancers (NSCLC) with high programmed cell death-ligand 1 (PD-L1) expression (≥ 50%) and second-line treatment regardless of PD-L1 status, though not all patients benefit from this approach. Much effort is ongoing to identify robust prognostic and predictive biomarkers of response to immune checkpoint inhibitors, overcoming PD-L1 that appears limited in its ability to discriminate patient candidates to this new class of anticancer agents. The purpose of this research study is to identify potential new biomarkers for immunotherapy in lung cancer. Fifty-three consecutive patients with advanced NSCLC treated with nivolumab were enrolled in the study. All the patients received a blood analysis looking for the relationship between different populations of baseline white blood cells and granulocytic myeloid-derived suppressor cells (Gr-MDSC) detected by flow cytometry, to identify and characterize patients with poor likelihood of benefit from nivolumab in NSCLC second-line setting, regardless of clinical feature and PDL1 expression. Univariate analysis showed that high baseline levels of Gr-MDSC and low baseline CD8/Gr-MDSC ratio are associated with significantly better (P = 0.02) response to immunotherapy treatment. Log-rank tests suggested a significant improvement in OS and PFS with high baseline levels of Gr-MDSC levels (≥ 6 cell/μl), low absolute neutrophil count (< 5840/μl), high eosinophil count (> 90 /μl), and NLR < 3. The multivariate analysis showed a statistically significant improvement for PFS (P = 0.003) and OS (P = 0.05) in favour of the identified good prognostic Gr-MDSC-linked asset group, compared with the poor prognosis group. The role of Gr-MDSC appears interesting as a potential biomarker in NSCLC patients receiving immune-checkpoint inhibitors. Further analyses are needed to confirmed and study in deep the role of these particular cells and their role in cancer response and progression during ICI therapy.

PS1169 CYTOKINE AND ARGINASE‐1 EXPRESSION DYNAMICS IN PATIENTS WITH CHRONIC MYELOID LEUKEMIA REVEALS EARLY MOLECULAR RESPONSE TO IMATINIB

Background:Imatinib remains as the preferred tyrosine kinase inhibitor (TKI) to treat chronic myeloid leukaemia (CML) patients. Besides its direct action targeting BCR‐ABL1, TKI therapy may also influence the anti‐tumour response. A broadly compromised immune system has been described at time of diagnosis supporting for a suppression of anti‐CML immune responses. These abnormalities include an expansion of the suppressive immune cell populations, myeloid derived suppressor cells (MDSCs) and regulatory T cells (Treg), and dysfunctional effector NK‐ and T‐immune responses. On TKI treatments, CML patients seem to re‐activate their immune system restoring the effector‐mediated immune surveillance.Aims:To describe TNF, IFNG, IL6, IL10, TGBF1, and ARG1 gene expression dynamic regarding molecular response to imatinib, since previous studies were designed by different approaches, criteria for timing and longitudinally studies are scare or missing.Methods:We analysed 106 peripheral blood samples of 64 CML patients, with multiple samples of 34 serially followed, and 26 of healthy donors. Were included patients at diagnosis (n = 23) and on imatinib 400 mg at 3 months (n = 41), 6 months (n = 42) classified according to the molecular response. An aliquot of the stored sample used to monitor BCR‐ABL1 level was retro‐transcribed to cDNA and TNF, IFNG, IL6, IL10, TGFB1 and ARG1 gene expression was quantified by PCR real time.Results:For patients at diagnosis, the expression of TNF, IFNG, IL6, IL10 and TGFB1 was significantly decreased when compared with healthy controls (Mann‐Whitney test p = 0.0336, p &lt; 0.0001, p = 0.0007, p &lt; 0.0001 and p = 0.0001, respectively); while ARG1 was significantly increased (Mann‐Whitney test p &lt; 0.0001). The level of ARG1 expression correlated with the level of IL10 and TGFB1 expressions (Spearman r = 0.54, p = 0.0368 and r = 0.74, p = 0.0057, respectively), and IL10 with TGFB1 expression (Spearman: r = 0.65, p = 0.0087). Once imatinib therapy was initiated, those patients who achieved an early molecular response (EMR, BCR‐ABL1 ≤10% at 3 months) significantly increased the expression of the pro‐inflammatory cytokines TNF and IL6 when compared with healthy controls, and the ARG1 expression decreased to control levels. At 6 months, the enhancement of TNF and IL6 was observed in either responders (BCR‐ABL1 &lt;1%) or non‐responders (≥1%), whereas the decrease of ARG1 expression was similar to control levels just in responders. At both 3 and 6 months of treatment, TNF correlated with IL6 expression only in responders (Spearman: r = 0.48, p = 0.0222; and r = 0.57, p = 0.0168, respectively). Furthermore, the longitudinal analysis between diagnosis and 3 months on therapy, those patients who achieve an EMR showed a statistically significant increase of 3 and 13 folds of TNF and IL6, respectively, and a significant 22‐fold decrease of ARG1 (Wilcoxon test: p = 0.0444, p = 0.0038 and p = 0.0094, respectively).Summary/Conclusion:Our results are in agreement with a significant immune suppression in CML patients at diagnosis, mediated by MDSCs and their influence on Tregs; also with a stimulatory effect on the immune system after imatinib initiation, especially in those who responded at 3 months. The dynamic of cytokines and, principally, ARG1 may play a role as an immune biomarker to monitor the response to TKI allowing the differentiation of optimal responders.

Effect of COPD on Inflammation, Lymphoid Functions and Progression-Free Survival during First-Line Chemotherapy in Advanced Non-small Cell Lung Cancer

Chronic obstructive pulmonary disease (COPD) is a common comorbidity of non-small cell lung cancer (NSCLC). COPD is characterized by systemic inflammation and lymphocyte dysfunction, mechanisms that are also known to accelerate progression of advanced (IIIB-IV) stage NSCLC. We aimed to find out whether COPD exerts an influence on tumor induced inflammatory and lymphoid responses and progression-free survival (PFS) after first-line treatment in advanced NSCLC. Patients suffering from NSCLC (n = 95), COPD (n = 54), NSCLC+COPD (n = 80) and healthy controls (n = 60) were included. PFS, neutrophil granulocyte and lymphocyte cell counts were recorded. Serum IFNγ, TNFα, VEGF concentrations were measured by using multiplex cytometric bead-based immunoassay. Prevalence of myeloid-derived suppressor cell populations (MDSC-s), and signs of T cell exhaustion were tested by using flow cytometry. Median PFS increased in the NSCLC+COPD group compared to NSCLC patients without COPD (7.4 vs 4.9 months, p < 0.01). NSCLC+COPD patients had 1.7 times (1.2–2.4) more likely to have longer PFS compared to NSCLC patients without COPD (Cox analysis, p < 0.01). Neutrophil cell counts, CRP, IFNγ and TNFα concentrations were all reduced in NSCLC+COPD (all p < 0.05 vs NSCLC). NSCLC+COPD was also associated with reduced serum IL-10 concentration and increased granzyme-B positive CD8 cell counts compared to NSCLC without COPD. The effects of VEGF and MDSC-s on systemic inflammation appeared to be blunted by COPD in patients suffering from advanced NSCLC. Concomitant COPD moderates tumor-induced inflammation and supports some effector lymphoid functions and thereby may be an independent positive predictive factor of longer PFS after first-line therapy in advanced NSCLC.

The role of 2-arachidonoylglycerol in the regulation of the tumor-immune microenvironment in murine models of pancreatic cancer

The pancreatic ductal adenocarcinoma (PDAC) microenvironment plays a critical role in the antitumor immune response. 2-arachidonoylglycerol (2-AG) exhibits a direct antitumor effect in various tumor models. However, the immunomodulatory effect of 2-AG on PDAC remains obscure. The aim of this study was to explore the tumor microenvironment response to 2-AG in pancreatic cancer. A PDAC orthotopic tumor model was used to investigate tumor proliferation and the population of immune cells in vivo, including dendritic cells (DCs), myeloid-derived suppressor cells (MDSCs), CD8+ T cells and CD4+ T cells. The effect of 2-AG on panc02 cell proliferation and DC2.4 cell maturation in vitro by mediating activation the typical cannabinoid receptors (CB1, CB2) was evaluated by flow cytometry and CCK8. The protein levels of P-STAT6, STAT6 and GADPH were measured by Western blotting.2-AG inhibited pancreatic cancer cell proliferation in tumor bearing mice and panc02 cell. Inhibition of proliferation was blocked by the CB1receptor antagonist (AM251) but not the CB2 receptor antagonist (AM630). In addition, 2-AG promoted DC phenotypic maturation and the production of proinflammatory cytokines by up-regulating p-STAT6. These effects were also blocked by AM251 but not AM630. Moreover, we also provide evidence that 2-AG administration induced the expansion of MDSCs in tumor bearing mice. However, no effect on the population of CD8+ T cells and CD4+ T cells was observed. Our findings support 2-AG exhibited direct antitumor effects via inhibiting pancreatic cancer proliferation and inducing DC phenotypic maturation, but also significantly promoted an immunosuppressive microenvironment via increasing the suppressive immune cell population of MDSCs.

The effects of a CRISPR/Cas9 IL-6 knockout in 4T1 mammary carcinoma cells on myeloid-derived suppressor cells (MDSCs) and Th17/Th22 cells

Abstract The 4T1 mouse mammary carcinoma cell line is syngeneic to Balb/c mice and is an effective tumor model to study the interactions between the immune system and metastatic breast cancer. The pro-tumor effects that myeloid-derived suppressor cell (MDSC) populations have on CD8+ T cells and Tregs are well known, but comparably less attention has been directed at the relationship between MDSCs and Type III immune Th17/Th22 cells in breast cancer. MDSCs and Th17/Th22 cells have been linked in other inflammatory settings, so in this study, we investigated MDSC, Th17 and Th22 cell interactions in 4T1 tumors, and the role of tumor-derived IL-6 in these interactions. IL-6 plays a key role in the induction of MDSCs under pathological conditions, as well as fate determining in Th maturation. Thus, the removal of IL-6 from 4T1 tumors allowed us to better characterize the source of these interactions. Populations of Monocytic (M)-MDSCs (CD11b+Ly-6G−Ly-6Chi), Polymorphonuclear (PMN)-MDSCs (CD11b+Ly-6G+Ly-6Clow), Th17 (CD3+CD4+RORγt+IFN-γ−IL-17A+IL-22−) and Th22 (CD3+CD4+IFN-γ−RORγt−IL-17A−IL-22+) cells were identified in peripheral blood, spleen, tumor and bone marrow (BM) of mice bearing wild-type and IL-6 knockout 4T1 tumors. Finally, we isolated 4T1 induced MDSCs (CD11b+Gr-1+)and cocultured them with healthy donors to investigate Th17 and Th22 population expansion. Our findings provide further insight into the interactions among MDSCs, Th17 and Th22 cells in a murine cancer model, which may inform immunotherapy efficacy and outcomes in human breast cancer.

Determinant roles of dendritic cell-expressed Notch Delta-like and Jagged ligands on anti-tumor T\xa0cell immunity

BackgroundNotch intercellular communication instructs tissue-specific T-cell development and function. In this study, we explored the roles of dendritic cell (DC)-expressed Notch ligands in the regulation of T-cell effector function.MethodsWe generated mice with CD11c lineage-specific deletion of Notch Delta-like ligand (Dll)1 and Jagged (Jag)2. Using these genetically-ablated mice and engineered pharmacological Notch ligand constructs, the roles of various Delta-like and Jagged ligands in the regulation of T-cell-mediated immunity were investigated. We assessed tumor growth, mouse survival, cytokine production, immunophenotyping of myeloid and lymphoid populations infiltrating the tumors, expression of checkpoint molecules and T-cell function in the experimental settings of murine lung and pancreatic tumors and cardiac allograft rejection. Correlative studies were also performed for the expression of NOTCH ligands, NOTCH receptors and PD-1 on various subsets of myeloid and lymphoid cells in tumor-infiltrating immune cells analyzed from primary human lung cancers.ResultsMice with CD11c lineage-specific deletion of Notch ligand gene Dll1, but not Jag2, exhibited accelerated growth of lung and pancreatic tumors concomitant with decreased antigen-specific CD8+T-cell functions and effector-memory (Tem) differentiation. Increased IL-4 but decreased IFN-γ production and elevated populations of T-regulatory and myeloid-derived suppressor cells were observed in Dll1-ablated mice. Multivalent clustered DLL1-triggered Notch signaling overcame DC Dll1 deficiency and improved anti-tumor T-cell responses, whereas the pharmacological interference by monomeric soluble DLL1 construct suppressed the rejection of mouse tumors and cardiac allograft. Moreover, monomeric soluble JAG1 treatment reduced T-regulatory cells and improved anti-tumor immune responses by decreasing the expression of PD-1 on CD8+Tem cells. A significant correlation was observed between DC-expressed Jagged and Delta-like ligands with Tem-expressed PD-1 and Notch receptors, respectively, in human lung tumor-infiltrates.ConclusionOur data show the importance of specific expression of Notch ligands on DCs in the regulation of T-cell effector function. Thus, strategies incorporating selectively engineered Notch ligands could provide a novel approach of therapeutics for modulating immunity in various immunosuppressive conditions including cancer.

Radiotherapy Both Promotes and Inhibits Myeloid-Derived Suppressor Cell Function: Novel Strategies for Preventing the Tumor-Protective Effects of Radiotherapy.

Cancer immunotherapies aimed at neutralizing the programmed death-1 (PD-1) immune suppressive pathway have yielded significant therapeutic efficacy in a subset of cancer patients. However, only a subset of patients responds to antibody therapy with either anti-PD-1 or anti-PD-L1 antibodies. These patients appear to have so-called “hot” tumors containing tumor-reactive T cells. Therefore, checkpoint blockade therapy may be effective in a larger percentage of cancer patients if combined with therapeutics that also activate tumor-reactive T cells. Radiotherapy (RT) is a prime candidate for combination therapy because it facilitates activation of both local antitumor immunity and antitumor immunity at non-radiated, distant sites (abscopal response). However, RT also promotes tumor cell expression of PD-L1 and facilitates the development of myeloid-derived suppressor cells (MDSC), a population of immune suppressive cells that also suppress through PD-L1. This article will review how RT induces MDSC, and then describe two novel therapeutics that are designed to simultaneously activate tumor-reactive T cells and neutralize PD-1-mediated immune suppression. One therapeutic, a CD3xPD-L1 bispecific T cell engager (BiTE), activates and targets cytotoxic T and NKT cells to kill PD-L1+ tumor cells, despite the presence of MDSC. The BiTE significantly extends the survival time of humanized NSG mice reconstituted with human PBMC and carrying established metastatic human melanoma tumors. The second therapeutic is a soluble form of the costimulatory molecule CD80 (sCD80). In addition to costimulating through CD28, sCD80 inhibits PD-1 suppression by binding to PD-L1 and sterically blocking PD-L1/PD-1 signaling. sCD80 increases tumor-infiltrating T cells and significantly extends survival time of mice carrying established, syngeneic tumors. sCD80 does not suppress T cell function via CTLA-4. These studies suggest that the CD3xPD-L1 BiTE and sCD80 may be efficacious therapeutics either as monotherapies or in combination with other therapies such as radiation therapy for the treatment of cancer.

A CD40 Agonist and PD-1 Antagonist Antibody Reprogram the Microenvironment of Nonimmunogenic Tumors to Allow T-cell–Mediated Anticancer Activity

In cancers with tumor-infiltrating lymphocytes (TILs), monoclonal antibodies (mAbs) that block immune checkpoints such as CTLA-4 and PD-1/PD-L1 promote antitumor T-cell immunity. Unfortunately, most cancers fail to respond to single-agent immunotherapies. T regulatory cells, myeloid derived suppressor cells (MDSCs), and extensive stromal networks within the tumor microenvironment (TME) dampen antitumor immune responses by preventing T-cell infiltration and/or activation. Few studies have explored combinations of immune-checkpoint antibodies that target multiple suppressive cell populations within the TME, and fewer have studied the combinations of both agonist and antagonist mAbs on changes within the TME. Here, we test the hypothesis that combining a T-cell-inducing vaccine with both a PD-1 antagonist and CD40 agonist mAbs (triple therapy) will induce T-cell priming and TIL activation in mouse models of nonimmunogenic solid malignancies. In an orthotopic breast cancer model and both subcutaneous and metastatic pancreatic cancer mouse models, only triple therapy was able to eradicate most tumors. The survival benefit was accompanied by significant tumor infiltration of IFNγ-, Granzyme B-, and TNFα-secreting effector T cells. Further characterization of immune populations was carried out by high-dimensional flow-cytometric clustering analysis and visualized by t-distributed stochastic neighbor embedding (t-SNE). Triple therapy also resulted in increased infiltration of dendritic cells, maturation of antigen-presenting cells, and a significant decrease in granulocytic MDSCs. These studies reveal that combination CD40 agonist and PD-1 antagonist mAbs reprogram immune resistant tumors in favor of antitumor immunity.

Abstract A176: Overcoming CD8 T-cell suppression in the tumor microenvironment

Abstract CD8 T-cell-mediated antitumor immunity is required for the control and elimination of tumors. However, tumors are able to overcome immune response resulting in immunosuppression, tumor growth, and metastatic spread. CD8 T-cells are controlled through a homeostatic network of positive and negative feedback loops. These negative signals are exploited by the tumor and associated suppressive cell populations in the tumor microenvironment. Immunotherapies targeted to receptors that control these negative signals, termed immune checkpoint inhibitors, have provided robust and durable responses to a number of cancers. Unfortunately, only a subset of patients will respond to current immunotherapies. This is due to the accruement of suppressive and inhibitory mechanisms employed by the tumor and its microenvironment. These include expression of inhibitory ligands, release of immune-suppressive factors, and the recruitment of suppressive cell populations. Furthermore, the administration of immune checkpoint inhibitors can enhance tumor acquisition of a more suppressive phenotype, diminishing immune responses through additional inhibitory pathways. We therefore propose that further CD8 T-cell-intrinsic negative regulators most likely exist, which can be targeted to improve antitumor responses. Here, we have used functional genomic approaches to identify pathways that can be targeted to advance anti-tumor responses, either in combination with anti-PD-1 or to overcome suppressive mechanisms employed by the tumor microenvironment. Using both in vivo and in vitro assays, we have identified both a novel target that provides an enhancement to CD8 T-cell effector function when in combination with PD-1 checkpoint blockade, and CD8 T-cell-intrinsic pathways that are modulated by the presence of suppressive cells associated with the tumor microenvironment. Citation Format: Adam N.R Cartwright, Peng Jiang, Assieh Saadatpour, Guo-Cheng Yuan, Shirley X. Liu, Kai W. Wucherpfennig. Overcoming CD8 T-cell suppression in the tumor microenvironment [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A176.

Abstract A138: CD39 increase on cytotoxic T-cell induced by myeloid-derived suppressor cell correlated with poor prognosis in patients with non-small cell lung cancer

Abstract Background: The factors in tumor microenvironment hinder T-cell activities against tumor cells. The major immunosuppressive cells in tumor sites are myeloid-derived suppressor cell (MDSC), tumor-associated macrophage (TAM), and regulatory T (Treg) cell, and the effector molecules released by those immunosuppressive cells also regulate T-cell activities. Therefore, in this study we examined the pattern of immunosuppressive cells in patients with non-small cell lung cancer. Then, we tested T-cell activities to verify whether the suppressive immune cell populations can influence T-cell activity by monitoring T-cell exhaustion markers. Since CD39 and CD73 expression on cytotoxic T-cell are known to be T-cell exhaustion markers, we analyzed CD39 and CD73 on CD8+ T-cells. Method: Baseline and one week after anti-PD-1 immunotherapy (pembrolizumab and nivolumab) blood samples (n=81) were collected (stage III and IV). For the correlation of suppressive immune cells with disease progression, baseline blood samples from the patients (n=59, stage I~IV) and healthy donors (n=21) were collected. Granulocytic-MDSC, Monocytic-MDSC, TAM, Treg, and CD39+ and CD73+ cytotoxic T-cell population from patients’ PBMC (n=81 and n=59) were analyzed by FACS Verse. For the suppressive assay, isolated T-cells were activated with anti-CD3 and anti-CD28 and then MDSC was co-cultured with T-cells for a week followed by Ki-67, CD39 and CD73 analysis by FACS Verse. Results: G-MDSC (p-value=0.0023), M-MDSC (p-value=0.0032), TAM ((p&amp;lt;0.001), and Treg (p&amp;lt;0.001) population were higher in advanced non-small cell lung cancer patients (stage III&amp;IV) compared with stage I and II patients or healthy donors. MDSC isolated from patient’s blood was co-cultured with activated T-cells from the same patient. After one week, T-cell activity was inhibited compared with T-cell alone (p &amp;lt; 0.001, E:T = 5:1, 10:1) confirming suppressive activity of MDSC against T-cells. CD39+CD8+ T-cells were also increased when co-cultured with MDSC. Low MDSC (p-value=0.02) and CD39+CD8+ T-cell population (p-value=0.043) in pre and post-anti-PD-1 immunotherapy and the population decreased group showed better overall survival compared to high and increased MDSC and CD39+CD8+ T-cell population group of the patients. Conclusion: G-MDSC and M-MDSC population increased as disease progressed and MDSC effectively suppressed T-cell activities and induced T-cell exhaustion. TAM and Treg population also increased in advanced non-small cell lung cancer patients. Increased MDSC and CD39+CD8+ T-cells correlated with poor prognosis in patients treated with anti-PD-1 immunotherapy. Therefore, high MDSC and increased CD39 expression on cytotoxic T-cell can be used as a prognostic biomarker for anti-PD-1 immunotherapy in patients with non-small cell lung cancer. Citation Format: Jiae Koh, Kyung Young Lee, Boram Kim, Mi Soon Kim, Hee Jin Cho, Jong-Mu Sun, Jin Seok Ahn, Keunchil Park, Myung-Ju Ahn. CD39 increase on cytotoxic T-cell induced by myeloid-derived suppressor cell correlated with poor prognosis in patients with non-small cell lung cancer [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A138.

Perivascular Cells and NADPH Oxidase Inhibition Partially Restore Hyperglycemia-Induced Alterations in Hematopoietic Stem Cell and Myeloid-Derived Suppressor Cell Populations in the Bone Marrow

Background and ObjectivesPatients suffer from long-term diabetes can result in severe complications in multiple organs through induction of vascular dysfunctions. However, the effects of chronic hyperglycemic conditions on hematopoiesis and the microenvironment in the bone marrow (BM) are not yet well understood.MethodsBM cells were harvested from femurs of mice and analyzed using flow cytometry. Human PVCs were cultured in serum-free α-MEM. After 24hrs, PVC-CM was collected and filtered through a 0.22 μm filter.ResultsIn this study, we showed that hyperglycemia alters hematopoietic composition in the BM, which can partially be restored via paracrine mechanisms, including perivascular cells (PVCs) and NADPH oxidase (NOX) inhibition in mice with streptozotocin-induced diabetes. Prolonged hyperglycemic conditions resulted in an increase in the frequency and number of long-term hematopoietic stem cells as well as the number of total BM cells. The altered hematopoiesis in the BM was partially recovered by treatment with PVC-derived conditioned medium (CM). Long-term diabetes also increased the number of myeloid-derived suppressor cells in the BM, which was partially restored by the administration of PVC-CM and diphenyleneiodonium (DPI), a NOX inhibitor. We further showed the downregulation of ERK and p38 phosphorylation in BM cells of diabetic mice treated with PVC-CM and DPI. This may be associated with dysfunction of hematopoietic cells and promotion of subsequent diabetic complications.ConclusionsOur data suggested that alterations in BM hematopoietic composition due to prolonged hyperglycemic conditions might be restored by improvement of the hematopoietic microenvironment and modulation of NOX activity.

Granulocytic myeloid-derived suppressor cell population increases with the severity of alcoholic liver disease.

Alcoholic liver disease (ALD) is a progressive liver disease that can cause a series of complications, including cirrhosis, liver failure and hepatocellular carcinoma. Granulocytic myeloid‐derived suppressor cell (gMDSC) populations have been observed to expand in various liver diseases and to inhibit innate and adaptive immunity in patients with liver disease. However, the characteristics of gMDSCs in patients with ALD have not been studied. We studied 24 healthy controls (HCs) and 107 patients with ALD and found an accumulation of gMDSCs in the peripheral blood of patients with alcoholic liver cirrhosis (ALC). Furthermore, ALC patients with a poor prognosis displayed a significant increase in peripheral gMDSCs and showed an increased capacity for arginase I production compared to HCs. In contrast, plasma arginase I levels in ALC patients were negatively correlated with total bilirubin and international normalized ratio, two key parameters of liver damage. Importantly, gMDSCs accumulated in the livers of ALC patients, and the frequency of liver gMDSCs significantly correlated with that of peripheral gMDSCs. In addition, gMDSC enrichment in vitro significantly inhibited the function of natural killer (NK) cells, perhaps preventing the NK‐induced apoptosis of hepatic stellate cells. In summary, increased peripheral and intrahepatic gMDSC populations are present in patients with ALC and may contribute to enhancing the severity of liver cirrhosis.

The Potential for Cancer Immunotherapy in Targeting Surgery-Induced Natural Killer Cell Dysfunction.

Natural Killer (NK) cells are granular lymphocytes of the innate immune system that are able to recognize and kill tumor cells without undergoing clonal selection. Discovered over 40 years ago, they have since been recognized to possess both cytotoxic and cytokine-producing effector functions. Following trauma, NK cells are suppressed and their effector functions are impaired. This is especially important for cancer patients undergoing the removal of solid tumors, as surgery has shown to contribute to the development of metastasis and cancer recurrence postoperatively. We have recently shown that NK cells are critical mediators in the formation of metastasis after surgery. While research into the mechanism(s) responsible for NK cell dysfunction is ongoing, knowledge of these mechanisms will pave the way for perioperative therapeutics with the potential to improve cancer outcomes by reversing NK cell dysfunction. This review will discuss mechanisms of suppression in the postoperative environment, including hypercoagulability, suppressive soluble factors, the expansion of suppressive cell populations, and how this affects NK cell biology, including modulation of cell surface receptors, the potential for anergy, and immunosuppressive NK cell functions. This review will also outline potential immunotherapies to reverse postoperative NK dysfunction, with the goal of preventing surgery-induced metastasis.

Knockout Tumor Microenvironment HO-1 Neutralizes Myeloid-Derived Suppressor Cells and Enhances the Antitumor Effect of PD-1 Inhibition in Murine Models of Acute Myeloid Leukemia

Background/Aims: Myeloid-derived suppressor cells (MDSCs) has been shown to be involved in tumor immune escape mechanisms and B cell-specific immunity in Acute myeloid leukemia (AML) patients. In our previous study shown that HO-1 protein have immunomodulatory activity including targeting of immune suppressor cells in the tumor microenvironment. Thus, we decided to assess whether HO-1 could enhance anti-PD-1 treatment and investigate those alterations in the immunosuppressive tumor microenvironment that contribute to the combined antitumor activity.Method: We utilized C57/BL6 (HO-1 knockout) mouse were radiation with 6.5GY once in a row to repress residual immunity. Malignant tumor HL-60 cells respectively (1×107 cells) per animal were injected subcutaneously into the right abdomen. The xenograft mouse models of AML were euthanised on the 14th day after treatment with a PD-1 inhibitor once a day (20 mg/kg). The tumor volumes were measured and calculated by ruler. Survival curve of individual groups was evaluated from the first day of treatment until death using Kaplan-Meier curves.Results: HO-1 gene knockout enhanced the antitumor effect of PD-1 inhibition in xenograft mouse models of AML by reducing tumor growth and increasing survival. HO-1 gene knockout inhibited the immunosuppressive function of both polymorphonuclear (PMN)- and monocytic-myeloid derived suppressor cell (M-MDSC) populations. Analysis of MDSC response to HO-1 gene knockout revealed significantly reduced arginase-1, iNOS, and COX-2 levels, suggesting potential mechanisms for the altered function. We also observed significant alterations in cytokine/chemokine release in vivo with a shift toward a tumor-suppressive microenvironment.Conclusions: Our results demonstrate that HO-1 gene knockout enhances the antitumor effect of PD-1 targeting through functional inhibition of MDSCs and a transition away from an immune-suppressive tumor microenvironment.[Key words] HO-1 gene knockout; PD-1 inhibitor; tumor microenvironment; MDSCs; AML DisclosuresNo relevant conflicts of interest to declare.

Protective immunity elicited by measles vaccine exerts anti-tumor effects on measles virus hemagglutinin gene-modified cancer cells in a mouse model.

Measles vaccine is widely used in China to prevent the measles virus (MV) infection. People immunized with measles vaccine can obtain long-term protective immunity. Measles virus surface glycoprotein hemagglutinin (H) can also induce MV-specific immune responses. However, little is known about whether the existence of the protective immune system against MV in the host can exert anti-tumor effects and whether the MV-H gene can serve as a therapeutic gene. We first vaccinated mice with measles vaccine, then inoculated them with MV-H protein-expressing tumor cells and observed the rate of tumor formation. We also treated mice with H protein-expressing tumor cells with measles vaccine and assessed tumor size and overall survival. Active vaccination using measles vaccine not only protected mice from developing tumors, but also eradicated established tumors. Measles vaccine elicited H-specific IFN-γ, TNF-α and granzyme B-producing CD8+ T cells and increased cytotoxic T lymphocyte (CTL) activity specific for H antigen, which provided a strong therapeutic benefit against H protein-expressing tumors. In addition, measles vaccine decreased the population of myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs). Our study demonstrated that tumor cells expressing H protein could activate the immune memory response against MV, which exerted specific anti-tumor effects, and indicated that the MV-H gene can be used as a potential therapeutic gene for cancer gene therapy.

Treg Fragility: A Prerequisite for Effective Antitumor Immunity?

Inhibitory checkpoint blockade has significantly improved patient response rate across numerous tumor types. However, most patients remain unresponsive to immunotherapy, suggesting that unappreciated mechanisms of resistance exist. The tumor microenvironment (TME) is unique and composed of many suppressive cell populations that inhibit antitumor immune responses, including regulatory T cells (Tregs). The TME is nutrient poor, acidic, and hypoxic, creating a challenging microenvironment for immune cells to function and survive. Tregs suppress a wide variety of cell populations through multiple mechanisms and are tasked with limiting tissue damage. Tregs are now considered to be a barrier to effective antitumor immunity. Systemic Treg depletion is not favored because of their critical role in maintaining immune homeostasis and preventing autoimmunity. Reducing Treg function specifically within the TME may provide a more effective, targeted approach to limit the immunosuppressive environment within the tumor without inducing systemic adverse consequences. Targeting molecules that cause Treg instability, characterized by loss of critical Treg transcription factors such as Foxp3, could result in conversion into cells that cause immune pathology, tissue damage, and subsequent autoimmune side effects. Interferon-γ (IFNγ) can cause intratumoral Treg "fragility," which results in loss of suppressive activity and increased IFNγ production without loss of Foxp3 expression and gross Treg "identity." We reviewed the impact Tregs have on the TME and vice versa, and their implications for responsiveness to cancer immunotherapy. We propose that the extent to which intratumoral Tregs develop a "fragile" phenotype following immunotherapy will predict and dictate responsiveness. Cancer Immunol Res; 6(8); 882-7. ©2018 AACR.